The use of stem cells in a variety of treatments and therapies is receiving growing attention. Stem cells can be used to repair or replace damaged or defective tissues and have broad clinical applications for treating a wide range of diseases.
Cell expansion systems can be used to grow stem cells, as well as other types of cells, both adherent and non-adherent. Adherent cells require a surface to attach to before the cells will grow and divide. Non-adherent cells grow and divide while floating in suspension.
Cell expansion systems provide nutrients to the growing cells and remove metabolites, as well as furnishing a physiochemical environment conducive to cell growth. Cell expansion systems are known in the art.
As a component of a cell expansion system, a bioreactor, or cell growth chamber, plays an important role in providing an optimized environment for the expanding cells. There are many types of bioreactors known in the art. Bioreactor devices include culture flasks, roller bottles, shaker flasks, stirred-tank reactors, air-lift reactors and hollow fiber bioreactors.
Once expanded cells in a bioreactor reach either confluence or the desired number of cells, they need to be harvested, and if further growth is desired, the cells need to be reseeded into the same or different bioreactor.
Regardless of which type of bioreactor device is used, to harvest adherent cells, the cells must first be removed from the surface they are growing on. In order to remove the adherent cells from the growth surface, the cells are initially washed to remove ions which inhibit trypsin (magnesium, calcium). Trypsin is then added to the washed cells to loosen them from the surface. Once the cells are loosened, they are removed from the membrane surface and processed to remove the trypsin either by washing the removed cells or spinning them down into a pellet, removing the surrounding fluid and suspending them in new growth media.
This procedure is easily done in open systems, such as culture flasks where the procedure takes place in a laminar flow hood, with the cells growing on flat plates. However, in closed systems using a hollow fiber bioreactor, the system is closed to the atmosphere. There is no easy way to add or remove fluids from the system, and ions contained in the growth media necessary for cell growth are lost from the cellular growth space due to ultrafiltration across the hollow fibers. The cells are therefore living in an environment surrounded by diluted media, (from the addition of fluid into the system from the initial washing of the cells) trypsin (from the trypsin used to loosen the cells from the membrane) and no ions in the media (calcium and magnesium for example) as a result of ultrafiltration.
These factors may contribute to the lack of cell growth of harvested adherent cells which are directly reseeded into a hollow fiber bioreactor.
Therefore, new reseeding protocols need to be developed for use in cell expansion systems using a hollow fiber bioreactor.